PROJECT 5 ? SUMMARY The goal of this proposal is to define the intracellular signaling pathways that contribute to the synthesis of phosphorylated phosphatidylinositols (phosphoinositides) in platelets, and to understand the role of phosphoinositides in platelet biology. Our recent research has focused on the synthesis of two phosphatidylinositol bisphosphates, PtdIns(4,5)P2 and PtdIns(3,5)P2. The hypotheses underlying this proposal are that the synthesis of PtdIns(4,5)P2 and PtdIns(3,5)P2 occurs within unique microdomains in platelets, and that PtdIns(4,5)P2 and PtdIns(3,5)P2 are each responsible for discrete aspects of platelet signaling and in vivo thrombus formation. We have studied the synthesis of PtdIns(4,5)P2 by the ?, ?, and ? isoforms of phosphatidylinositol phosphate 5-kinase I (PIP5KI), and found clear evidence that spatially distinct pools of PtdIns(4,5)P2 exist in platelets and in megakaryocytes. Moreover, the loss of either PIP5KI? or PIP5KI? leads to impaired in vivo thrombosis, but for different reasons. The pool of PtdIns(4,5)P2 that is synthesized by PIP5KI? is required to form second messengers and to extend lamellipodia. In contrast, PtdIns(4,5)P2 synthesized by PIP5KI? is vital for the membranecytoskeletal dynamics and allows for stable adhesion that is resistant to shear forces. We have also found that PtdIns(3,5)P2, which is synthesized by PIKFyve, regulates the biogenesis of platelet lysosomes. Deletion of PIKFyve specifically in murine platelets leads to a platelet lysosome disorder, which remarkably is associated with inflammation and accelerated thrombus formation in vivo. Taken together, these results support the hypothesis that the synthesis of particular phosphoinositides within discrete platelet subcellular microdomains regulates specific spatially distinct intracellular signaling pathways. The proposed experiments are designed to comprehensively and systematically study how PtdIns(4,5)P2 and PtdIns(3,5)P2 production regulates the platelet cytoskeleton and lysosomes. We will also study their effect on stable platelet adhesion in vivo. In Aim 1, we will determine how PtdIns(4,5)P2 regulates platelet membrane-cytoskeletal dynamics. In Aim 2, we will examine the spatial regulation of PtdIns(4,5)P2 synthesis. In the final Aim, we will formally analyze how PtdIns(3,5)P2 regulates the biology of the platelet lysosome and its contribution to in vivo thrombus formation.